1. Technical Field
The present invention relates to the field of the optical analysis (imaging and spectroscopy) of luminescent or optically diffusing particles.
It relates more particularly to an imaging system for analysing fluorescent particles in a sample, comprising a confocal or large-field microscopy device and comprising a support in contact with at least part of this sample. This imaging system finds its applications mainly in the implementation of a fluorescence correlation spectroscopy system, as well as the observation and study, in a biological medium, of the points of contact between a cell and the support of this imaging system.
It also relates to a sample support intended to equip such an imaging system, as well as to a method of analysing fluorescent molecules in a sample using such an imaging system.
2. Prior Art
A general technical problem in this field relates to the detection of individual molecules, whether by imaging in general or more specifically by fluorescence correlation spectroscopy (FCS). The molecules that are to be detected are fluorescent molecules, situated for example in a biological medium.
Detection of a single molecule generally requires the use of an ultrapure solvent (for example pure water) in order to reduce as far as possible the background noise due to the environment. However, these conditions of purity of the solution are never obtained in the case of experiments in an in vivo biological medium, for which the signal to noise ratio is considerably impaired by the existence of a background noise, mainly because of the autofluorescence phenomenon in these cells, as well as the Raman diffusion of the components and the cell environment.
This background noise depends on the size of the observation volume: the larger the observation volume, the greater the background noise, and vice versa.
Moreover, detecting individual molecules requires having a very large quantity of molecules in the observation volume, ideally a single molecule. Such observation conditions can be obtained using sufficiently diluted fluorophor solutions, but also by reducing the detection volume as far as possible.
Moreover, under conventional FCS conditions (i.e. on the basis of a confocal microscope) it is not possible to detect chemical species present at concentrations exceeding around 100 nanomols per liter (>100 nM). However, a large number of biological processes, such as enzymatic reactions, the expression of proteins or the action of medications, occur at concentrations of around 1 to 10 μM, or even more. Under these conditions, where the concentration of the molecules of interest becomes large, it then appears also necessary to considerably reduce the size of the observation volume, so as to make “visible” the fluctuations in the fluorescence signal and thus carry out the FCS. The same applies with the measurement of the diffusion time Td. This is because those greater than a few hundreds of microseconds (>100 ms) are very difficult to measure by conventional FCS, mainly because of the photobleaching of the fluorescent sensors. Many processes, such as the diffusion of membrane proteins, or diffusion in dense heterogeneous media such as collagen, may take place very slowly. A significant reduction in the size of the observation volume makes it possible to access these extremely slow phenomena more easily.
All these constraints are therefore aimed at reducing the size of the observation volume, which also makes it possible to eliminate the background noise.
To achieve this aim, a confocal microscope may be used, advantageously having a lens with a large numerical aperture.
This is because the image of any object is limited by optical diffraction. The limit of resolution is directly correlated with the diffraction (diffraction limit is spoken of). This depends on the illumination wavelength (λ) and the numerical aperture of the optical system (ON), and is equal to approximately λ/ON. Under these conditions, in the visible range, the optimum lateral resolution (perpendicular to the optical axis of the microscope) in traditional microscopy is around 300 to 400 nm. However, the phenomenon of diffraction also limits the axial resolution (i.e. along the optical axis) of the microscope. Confocal microscopy constitutes at the present time the most conventional tool for tending towards this limit imposed by diffraction. It makes it possible to reduce the observation volume in the three directions in space. This technique is based solely on the use of a pinhole filter in detection. The observation volume limited by diffraction then has an elliptical form and is elongate along the optical axis. With lenses with a large numerical aperture (ON>1.2) the confocal observation volume is usually between 0.2 and 1 μm3.
Under these conventional conditions (i.e. on the basis of a confocal microscope), FCS does not make it possible to detect chemical species present in concentrations exceeding around 100 nanomols per liter, nor to detect diffusion times greater than a few hundreds of milliseconds.
Consequently it appears necessary to reduce the size of the observation volume beyond what is offered by a microscope according to the prior art, so as to make the fluctuations in the fluorescence signal “visible” and thus to achieve an FCS measurement with greater precision.
Several solutions are known for addressing this problem of individual detection of molecules in a biological medium.
A first solution, a so-called STED technique, standing for “stimulated emission depletion”, consists of using two femtosecond (or picosecond) laser sources that are synchronised. This near-field technique is based on an inhibition of the fluorescence signal via a computing relaxation method: stimulated emission. Stimulated emission is used to “trim” the periphery of the focusing spot of the exciting laser illumination, thus reducing the lateral and axial dimensions of the confocal volume.
Nevertheless, the complexity relating to the use of this type of light source and to the use thereof in microscopy makes this technique complex.
A second solution, a so-called STORM technique, standing for “stochastic optical reconstruction microscopy”, or PALM standing for “photoactivated localisation microscopy”, is based on a very precise localisation of the fluorescence signal of individual molecules observed by means of a conventional large-field microscope. Even if the image in far field of a single molecule is limited by diffraction (200-300 nm laterally and 600-800 nm axially), it is always possible to find the precise position of the molecule (i.e. the central position of the spot limited by diffraction). A new image representing the exact positions of the molecules can then be constructed, which makes it possible to display membrane proteins with very great precision.
However, this solution has the drawback of requiring excessively long acquisition times for envisaging dynamic studies, such as movements of proteins for example.
Thus, a certain number of novel techniques for reducing the confocal observation volume have been proposed. These techniques make it possible to carry out local spectroscopy based on the analysis of the fluctuations of the fluorescence signal of individual molecules by FCS.
Fluorescence correlation spectroscopy (FCS) is based on the evaluation of the temporal autocorrelation function GAC(T) of the fluorescence signal F(t) emitted by molecules that pass through the optical detection volume:
                                                        G              AC                        ⁡                          (              τ              )                                =                                                    〈                                                      F                    ⁡                                          (                      t                      )                                                        ⁢                                      F                    ⁡                                          (                                              t                        +                        τ                                            )                                                                      〉                                                              〈                                      F                    ⁡                                          (                      t                      )                                                        〉                                2                                      =                                                            〈                                      δ                    ⁢                                                                                  ⁢                                          F                      ⁡                                              (                        t                        )                                                              ⁢                    δ                    ⁢                                                                                  ⁢                                          F                      ⁡                                              (                                                  t                          +                          τ                                                )                                                                              〉                                                                      〈                                          F                      ⁡                                              (                        t                        )                                                              〉                                    2                                            +                              l                ⁢                                                                  ⁢                where                                                    ⁢                                  ⁢                              δ            ⁢                                                  ⁢                          F              ⁡                              (                t                )                                              =                                    F              ⁡                              (                t                )                                      -                          〈                              F                ⁡                                  (                  t                  )                                            〉                                                          (        1        )            
δF(t) represents the fluctuations of the fluorescence signal (F(t) with respect to the mean signal <F(t)>.
According to equation (1), FCS is sensitive only to the fluctuations in the fluorescence signal, which are mainly related to the fluctuations in concentration of the molecules in the volume sounded. Nevertheless, these fluctuations may have several different origins and the physical processes that give rise to them occur to different timescales, FIG. 4.
In short times, typically less than 1 the change in GAC(T), denoted G(T) in FIG. 4, is mainly governed by the internal dynamics of the fluorescent molecule (passage through the triplet state, rotation, etc.).
At long times, the physical process that governs the change in GAC(T) is diffusion (Brownien motion or molecule flow). Then the amplitude of the autocorrelation function is inversely proportional to the number of molecules (<N>) contained in the observation volume; the decay time of GAC(T) corresponds for its part to the average time that a molecule takes for diffusing through this volume. This time is called the diffusion time (denoted Td in FIG. 4).
To this it is also necessary to add the chemical reactions that may occur in the solution and modify the dynamics of the molecule studied (photobleaching, quenching, chemisorption, etc.).
The evaluation of <N> and of Td constitute the two types of measurement that offer a major interest in biophysics. This is because, after a first calibration step in order to estimate the size of the volume sounded, it then becomes possible for a person skilled in the art to deduce from <N> and Td the diffusion coefficient D and the concentration of the biomolecules of interest.
Among these recent techniques based on FCS, a third solution consists of using a microscope of the SNOM type (scanning near-field optical microscope), which is based on the use of a near-field optical microscope using evanescent waves. Local illumination of the sample takes place for example by means of a stretched metallised optical fibre. A “nanosource” of light is then obtained at the end of the fibre. Consequently, only the molecules present at the end will be excited.
Nevertheless, this technique proves equally complex to implement in a biological medium, for reasons' similar to those evoked in relation to the second solution above (STORM).
Another so-called nanohole technique, able to adapt to conventional fluorescence, makes it possible to reduce the size of the observation volume laterally and axially (with reference to the optical axis of the microscope), and to amplify the fluorescence signal. For this purpose holes of nanometric size are produced in a metal film. The excitation then remains confined in the hole.
The latter technique does not however make it possible to image an object in its entirety, such as living cells. The metal layer may modify the adhesion of the cells and in particular prevent any observation by means of a usual biology technique (phase contrast, DIC, RICM).
Thus, it appears that no solution of the prior art makes it possible to significantly reduce the dimensions of the observation volume (typically a few attoliters) in order to achieve local fluorescence spectroscopy and/or imaging, while making it possible to image, in a biological medium, a set of living cells with very low axial resolution (less than 10 nm).
In other words, at the present time there exists no simple solution to be implemented that can be compatible with conventional fluorescence observation, in order to avoid reconstructing a new FCS spectroscopy system while offering a reduction in the observation volume.